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Protein analysis and characterization

Expression of SP1 related proteins in P. euphratica plants growing in hydroponics (lab exp.) and under greenhouse (pots) salt stress conditions was recorded. Six EST sequences homologous to the P. tremula SP1 protein were identified in the P. euphratica EST database POPEST. All the ESTs have exactly the same ORF showing 70.5% similarity and 61% identity to P. tremula SP1. SP1 proteins were extracted from leaves of P. euphratica plants and sequenced. The sequence shows 100 % homology to P. tremula SP1. Internal sequencing of SP1-related protein from leaves of P. euphratica plants growing under greenhouse conditions showed 100 % homology to P. tremula SP1.

Significant increment in SP1 levels was not observed upon salt stress in leaves, shoots or roots. Polyclonal anti-SP1 antibodies (P. tremula) were affinity purified against the P. euphratica SP protein. Western blot analysis revealed the specificity of the affinity purified antibody and are being used for sub-cellular in situ localization (immuno-gold labelling) of SP1 proteins in plant tissue exposed to salt stress. Preliminary results indicate specific accumulation of P. euphratica SP1 in cell membranes in response to salt stress. sp1 transcript was down-regulated under stress conditions in both P. tremula and P. euphratica, but was more pronounce at increasing salt concentrations in P. euphratica.

Hybridization of RNA extracted from salt experiments (hydroponics) was carried out on micro-arrays slides containing 350 stress related genes previously selected by all the participants. Among the analyzed genes, 3% were up-regulated on P. tremula plants in 150mM NaCl while no differences were observed in P. euphratica plants. On the other hand, 6% were severely down-regulated in P. tremula when plants grown in 150mM NaCl. Further experiments will be done as biological and technical repeats.

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